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Changes in the amounts and localization of protein kinase C (PKC) isoforms occur in galactosaemic lens epithelial cells. A link between PKC changes and myo-inositol depletion has been suggested. Raf-1, a component of a Ras pathway, is a substrate for PKC. Raf-1 levels were measured in galactosaemic lens epithelial cells grown with or without myo-inositol. Raf-1 levels were measured by densitometric scanning of Western blots from cells grown with or without 40 mmol/l galactose or 40 mmol/l galactose plus 1.0 micromol/l myo-inositol for 1, 3, 5 or 7 days. Scans were compared to those for PKCalpha, an isoform of PKC and to 14-3-3, a protein which binds to Raf-1. Cell growth was quantitated by thymidine incorporation. Raf-1 levels were decreased in bovine lens epithelial cells after 3, 5 or 7 days (33% of control) of growth in 40 mmol/l galactose. Addition of 1 micromol/l myo-inositol reversed this decrease at day 3, but not after 5 or 7 days of growth in 40 mmol/l galactose. PKCalpha and 14-3-3 levels were not affected by galactose. The decrease in Raf-1 was not a result of cell growth as measured by thymidine incorporation. These results suggest that Raf-1 levels are decreased during galactosaemia. This was only partially reversed by the addition of myoinositol.

To identify the role of PKC-gamma in control of and phosphorylation of connexin 46 (Cx46) in the lens cortex.The association between PKC-gamma and Cx46 was determined by co-immunoprecipitation from whole lens. Phosphorylation of Cx46 and activity of PKC-gamma were determined using Western blots, PKC activity assays, and inhibition of PKC activity by addition of isoform-specific PKC pseudosubstrate inhibitors.Co-localization of PKC-gamma and Cx46 was observed in the bow regions and cortical regions of rat lens. PKC-gamma was not observed in the nuclear region and Cx46 was not observed in the epithelial layer. PKC-alpha was not found in lens cortex or nuclear regions. PKC-gamma could be co-immunoprecipitated with Cx46 from lens cortical regions. Cx46 was phosphorylated on both serine and threonine. No tyrosine phosphorylation was observed. The PKC-gamma specific pseudosubstrate inhibitor caused a 73% inhibition of serine phosphorylation on Cx46 at 1 microM, and, 36% inhibition of threonine phosphorylation at the same concentration. Inhibition of phosphorylation of Cx46 with PKC-alpha pseudosubstrate inhibitor was not observed.PKC-gamma may phosphorylate Cx46, primarily on serine in whole lens. A role for PKC-gamma in control of lens cortical gap junctions is suggested.

The overexpression of PKCalpha or PKCgamma for extended periods of time causes the formation of lentoid bodies in the N/N 1003A rabbit lens epithelial cell line. To determine how differentiated the lentoid bodies are, we have looked for alphaA-, alphaB-, beta-, and gamma-crystallin levels in lentoid bodies after 4 and 8 weeks of lentoid body development.Cells overexpressing PKCalpha or PKCgamma were plated in 6 well plates and were allowed to form lentoid bodies for up to 8 weeks. Lentoid bodies were fixed and stained with PKCalpha or PKCgamma antibodies along with either alphaA-, alphaB-, beta-, or gamma-crystallin antisera and viewed under a confocal microscope. Lentoid bodies were harvested in lysis buffer and homogenized. Fifty micrograms of protein per lane was loaded onto an SDS-PAGE gel and the bands transferred onto nitrocellulose. The blot was probed with either alphaA-, alphaB-, beta-, or gamma-crystallin antibodies for 12 h. Total RNA from lentoid bodies was isolated and 5 microg of total RNA was transcribed to first-strand cDNA. The PCR products were analyzed by 2% agarose gel electrophoresis.alphaB-crystallin was present in normal N/N 1003A cells and the lentoid bodies formed from PKCalpha and PKCgamma overexpression. alphaA-crystallin was only detectable in lentoid bodies after PKCalpha or PKCgamma overexpression. RT-PCR was able to detect beta-crystallin expression while the Western blot analysis and immunocytostaining detected small amounts of beta-crystallin protein. No gamma-crystallin expression was noted in these lentoid bodies.Overexpression of PKCalpha or PKCgamma in the N/N 1003A cell line induced lentoid body formation. These lentoid bodies expressed not only alphaB-crystallin but alphaA- and beta-crystallin. These results suggest a role for PKCs in lens epithelial cell differentiation to a fiber cell.

Trichosanthin, from Trichosanthes kirilowii is a member of the Cucurbitaceae family. It has been reported to have anti-cancer activity. The effects of Trichosanthin peptides (15 amino acids in length) on ConA stimulated incorporation of 3[H] thymidine into cultured, primary mouse spleen cells and L1210 cells were determined. Both spleen cells and L1210 cells were plated at 7.5 x 10(5) cells/ml in a microtiter plate. Different concentrations of the peptide (0.5, 5, 25, and 50 micrograms/ml) and ConA (1 microgram/ml) were added with serum free media, and half of the samples were labeled with 1 microCi/well of 3[H] thymidine. The additional half of the cell samples were used to determine cell number and % viability. After 24 hours incubation, the N-terminal peptides (amino acid residues #1-15 and 16-30) caused increases in ConA stimulated incorporation of 3[H] thymidine into normal spleen cells at low concentrations of the peptides (5 micrograms/ml). The viability of spleen cells and L1210 cells were not affected by these peptides at 5 micrograms/ml. These N-terminal peptides (#1-15 and 16-30) were tested for in vivo anti-tumor activity. There was a delay of tumor formation in the treated vs control group. The results suggest that N-terminal sequences of trichosanthin have anti-tumor activity.

In rod outer segments the light activation of cGMP phosphodiesterase (PDE alpha beta gamma 2) is accomplished by removal of the gamma inhibitory subunit (PDE gamma) from the PDE alpha beta catalytic subunits. A light activation of the inositol signaling pathway also occurs, but there is little information linking these two signal transduction pathways. Here we report that protein kinase C (PKC) purified from bovine rod outer segment phosphorylates the bovine PDE gamma with incorporation of 0.9 +/- 0.1 mol of phosphate/mol of PDE gamma. Phosphorylation of PDE gamma increases its ability to inhibit PDE alpha beta catalytic activity (trypsin-activated PDE, tPDE) with an IC50 for phosphorylated PDE gamma of 26 +/- 4 pM and an IC50 of 60 +/- 5 pM for unphosphorylated PDE gamma. Inhibition of tPDE by PDE gamma is characterized by two values of Kd, Kd1 = 34 pM and Kd2 = 760 pM. Phosphorylation of PDE gamma by PKC eliminates the functional heterogeneity of the PDE gamma population resulting in a single value of Kd = 23 pM. Free PDE gamma (without PDE alpha beta catalytic subunits) is a better substrate for PKC than PDE gamma in a complex with PDE alpha beta. Phosphorylation of free PDE gamma by PKC is characterized by a value of Vmax = 1,550 +/- 148 units/mg (Km = 21.0 +/- 1.9 microM). In contrast, phosphorylation of PDE gamma in PDE alpha beta gamma 2 complex has two values of Vmax, Vmax1 = 0.3 +/- 0.1 units/mg of PDE gamma (Km1 = 0.4 +/- 0.2 microM) and Vmax2 = 0.7 +/- 0.2 units/mg of PDE gamma (Km2 = 4.6 +/- 0.9 microM). ROS PKC phosphorylates Thr35 in PDE gamma. We have previously reported (Morrison, D. F., Rider, M. A., and Takemoto, D. J. (1987) FEBS Lett. 222, 266-270; Lipkin, V. M., Udovichenko, I. P., Bodarenko, V. A., Yurovskaya, A. A., Telnykh, E. V., and Skiba, N. P. (1990) Biomed. Sci. (Lond.) 1, 305-308) that the central fragment of PDE gamma (24-45) is responsible for binding to PDE catalytic subunits. The new data suggests that this region of PDE gamma also includes the site for phosphorylation by PKC and that phosphorylation increases the ability of PDE gamma to inhibit PDE catalytic activity. This altered regulation of visual transduction may play a role in desensitization or light adaptation.

Retinal rod outer segment phosphodiesterase (PDE) consists of two similar catalytic subunits (alpha and beta) and two identical inhibitory subunits (gamma 2). A trypsin-activated soluble PDE exhibiting the ability to be reinhibited by PDE gamma was shown by peptide antisera to retain both N and C termini. Synthetic peptides corresponding to residues 16-30, 78-90, 389-403, and 535-563 of PDE alpha used in a PDE activity assay with trypsin-activated PDE partially prevented inhibition by exogenous PDE gamma; however, only competitions by peptides 16-30 and 78-90 (corresponding to PDE alpha 16-30 and 78-90) were concentration-dependent below 100 nmol of peptide. Binding studies using radio-immunoassays and PDE alpha peptides confirmed that peptides 16-30 and 78-90 (corresponding to PDE alpha 16-30 and 78-90, respectively) were able to bind PDE gamma. Additionally, peptides corresponding to the PDE alpha region 453-534 bound PDE gamma in the binding assay. This suggests that several regions on PDE alpha interact with the PDE gamma inhibitor. While some regions may be involved in binding to PDE gamma, other sites may be involved in PDE gamma inhibition of catalytic activity. Our results suggest that the major regions of PDE alpha that interact with PDE gamma reside within the N terminus (16-30 and 78-90), with weaker interaction regions within or near the hypothesized catalytic domain (453-563). Sequence analysis of three retinal phosphodiesterases (rod outer segment alpha, beta, and cone outer segment alpha') revealed the highest region of dissimilarity in the N and C termini.

The in vivo antitumor activity of a crude extract from the bitter melon (Momordica charantia) was determined. The extract inhibited tumor formation in CBA/H mice which had been given i.p. injections of 1.0 X 10(5) CBA/Dl tumor cells (77% of the untreated mice with tumors versus 33% of the treated mice with tumors after 6 weeks). The extract also inhibited tumor formation in DBA/2 mice which had been given i.p. injections of either 1 X 10(5) P388 tumor cells (0% of untreated mice survived after 30 days versus 40% survival of the treated mice) or 1 X 10(5) L1210 tumor cells (0% survival of untreated mice versus 100% of treated mice after 30 days). The in vivo antitumor effect required both the prior exposure of tumor cells to the extract (2 hr) in vitro and i.p., biweekly injections of the extract into the mice. The optimum dose for tumor inhibition (8 micrograms protein, biweekly, i.p.) was not toxic to mice for at least 45 days of treatment. This same treatment caused a marked enhancement of C3H mouse thymic cell response to concanavalin A in vitro. When compared to the untreated control mice, the bitter melon-injected animals exhibited a 4-fold-higher incorporation of tritiated thymidine into trichloroacetic acid-precipitable material after 48 hr of exposure to 50 micrograms of concanavalin A. Nylon wool-purified spleen cells from these same bitter melon-treated mice exhibited an enhanced mixed lymphocyte reaction when exposed to irradiated P388 stimulator cells (186% of the untreated control mice). These data indicate that in vivo enhancement of immune functions may contribute to the antitumor effects of the bitter melon extract.

Cyclic GMP is thought to be involved in lymphocytic cell proliferation and leukemogenesis. In general, the nucleotide is elevated in leukemic vs. normal lymphocytes and changes have been reported to occur during remission and relapse of this disease. Although the cA/cG ratios are higher for normal lymphocytes the basal guanylate cyclase (EC 4.6.1.2) activities do not correlate with altered cyclic GMP levels. The crude guanylate cyclases display classical Michaelis-Menten kinetics with Kms for Mn-GTP of 463 mumol/l and 20-90 mumol/l for normal and leukemic lymphocytes, respectively. An extract from the bitter melon (Momordica charantia) preferentially inhibits the soluble guanylate cyclase from leukemic lymphocytes. This inhibition correlates with its preferential cytotoxic effects for these same cells. Analyses of nine other cytotoxic plant extracts revealed that only an extract from the Lawson's cypress, Chamaecyparis lawsonianna, exhibits comparable cytotoxicity and guanylate cyclase inhibition levels.

Cyclic nucleotide phosphodiesterase activities were examined in lymphocytes from 12 transformed human B cell lines, two T cell lines, six patients with lymphocytic leukemia, and 10 normal donors. A consistent difference bwtween cells from the normal and leukemic state was observed. The cyclic AMP phosphodiesterase activity from normal lymphocytes is inhibited greater than 80% by muM cyclic GMP while this concentration of nucleotide has little or no effect on the enzyme from transformed lymphocytic cell lines or from lymphocytic cells of leukemia patients. The reported lack of cyclic GMP phosphodiesterase in human lymphocytes from several sources is confirmed. The apparent absence of a cyclic GMP degradation mechanism and of cyclic GMP control of cyclic AMP hydrolysis may be related to defective lymphocyte growth control.

There is considerable evidence which suggests that the gamma-subunit of cGMP phosphodiesterase (PDE gamma) is a multifunctional protein which may interact directly with both the catalytic subunits of PDE (PDE alpha beta) and the alpha-subunit of transducin (T alpha) (Whalen, M., and Bitensky, M. (1989) Biochem. J. 259, 13-19; Griswold-Prenner, I., Young, J. H., Yamane, H. K., and Fung, B. K.-K. (1988) Invest. Ophthalmol.Visual Sci. 29, (Suppl.) 218). To determine the region of interaction between the multifunctional PDE gamma and T alpha, and to determine the significance of this interaction, peptides corresponding to various regions of PDE gamma were synthesized and tested for their ability to inhibit the GTPase activity of T alpha. One of these peptides, PDE gamma-3 (bovine amino acid residues 31-45), inhibited the GTPase activity of T alpha with an I50 of 450 microM. The peptide (PDE gamma-3) was found to inhibit the GTPase activity of T alpha by inducing the binding of transducin to the rod outer segment membrane and by altering the GTP/GDP exchange. Analogs of PDE gamma-3 were synthesized to determine the required structure of the PDE gamma-3 region needed for the interaction of PDE gamma with T alpha. The results of these studies indicated that the removal of the positively charged amino acids or any of the potential hydrogen-bonding amino acids increased the I50 for the inhibition of the GTPase activity of T alpha Substitution of the hydrophobic amino acids had no effect. These results indicate the hydrophilic interactions may be essential for the binding of PDE gamma to T alpha and for the inhibition of the GTPase activity of T alpha by PDE gamma. The observed effects of PDE gamma-3 on T alpha and on PDE suggest that PDE gamma is a multifunctional protein which may play more than one role in the deactivation of the retinal transduction cascade.