Enlargement of the WHO international repository for platelet transfusion-relevant bacteria reference strains.

BACKGROUND AND OBJECTIVES: Interventions to prevent and detect bacterial contamination of platelet concentrates (PCs) have reduced, but not eliminated the sepsis risk. Standardized bacterial strains are needed to validate detection and pathogen reduction technologies in PCs. Following the establishment of the First International Reference Repository of Platelet Transfusion-Relevant Bacterial Reference Strains (the 'repository'), the World Health Organization (WHO) Expert Committee on Biological Standardisation (ECBS) endorsed further repository expansion. MATERIALS AND METHODS: Sixteen bacterial strains, including the four repository strains, were distributed from the Paul-Ehrlich-Institut (PEI) to 14 laboratories in 10 countries for enumeration, identification and growth measurement on days 2, 4 and 7 after low spiking levels [10-25 colony-forming units (CFU)/PC bag]. Spore-forming (Bacillus cereusPEI-B-P-07-S, Bacillus thuringiensisPEI-B-P-57-S), Gram-negative (Enterobacter cloacaePEI-B-P-43, Morganella morganiiPEI-B-P-74, PEI-B-P-91, Proteus mirabilisPEI-B-P-55, Pseudomonas fluorescensPEI-B-P-77, Salmonella choleraesuisPEI-B-P-78, Serratia marcescensPEI-B-P-56) and Gram-positive (Staphylococcus aureusPEI-B-P-63, Streptococcus dysgalactiaePEI-B-P-71, Streptococcus bovisPEI-B-P-61) strains were evaluated. RESULTS: Bacterial viability was conserved after transport to the participating laboratories with one exception (M. morganiiPEI-B-P-74). All other strains showed moderate-to-excellent growth. Bacillus cereus, B. thuringiensis, E. coli, K. pneumoniae, P. fluorescens, S. marcescens, S. aureus and S. dysgalactiae grew to >106 CFU/ml by day 2. Enterobacter cloacae, P. mirabilis, S. epidermidis, S. bovis and S. pyogenes achieved >106 CFU/ml at day 4. Growth of S. choleraesuis was lower and highly variable. CONCLUSION: The WHO ECBS approved all bacterial strains (except M. morganiiPEI-B-P-74 and S. choleraesuisPEI-B-P-78) for repository enlargement. The strains were stable, suitable for spiking with low CFU numbers, and proliferation was independent of the PC donor.

Prognostic value of tumor-infiltrating lymphocytes differs depending on histological type and smoking habit in completely resected non-small-cell lung cancer.

BACKGROUND: T-cell infiltration in tumors has been used as a prognostic tool in non-small-cell lung cancer (NSCLC). However, the influence of smoking habit and histological type on tumor-infiltrating lymphocytes (TILs) in NSCLC remains unclear. PATIENTS AND METHODS: We evaluated the prognostic significance of TILs (CD4+, CD8+, CD20+, and FOXP3+) according to histological type and smoking habit using automatic immunohistochemical staining and cell counting in 218 patients with NSCLC. RESULTS: In multivariate survival analyses of clinical, pathological, and immunological factors, a high ratio of FOXP3+ to CD4+ T cells (FOXP3/CD4) [hazard ratio (HR): 4.46, P < 0.01 for overall survival (OS); HR: 1.96, P < 0.05 for recurrence-free survival (RFS)] and a low accumulation of CD20+ B cells (HR: 2.45, P = 0.09 for OS; HR: 2.86, P < 0.01 for RFS) were identified as worse prognostic factors in patients with adenocarcinoma (AD). In non-AD, a low number of CD8+ T cells were correlated with an unfavorable outcome (HR: 7.69, P < 0.01 for OS; HR: 3.57, P < 0.02 for RFS). Regarding smoking habit in AD, a high FOXP3/CD4 ratio was poorly prognostic with a smoking history (HR: 5.21, P < 0.01 for OS; HR: 2.38, P < 0.03 for RFS), whereas a low accumulation of CD20+ B cells (HR: 4.54, P = 0.03 for OS; HR: 2.94, P < 0.01 for RFS) was confirmed as an unfavorable factor in non-smokers with AD. CONCLUSIONS: A low number of CD8+ T cells in non-AD, a high FOXP3/CD4 ratio in smokers with AD, and a low number of CD20+ B cells in non-smokers with AD were identified as independent unfavorable prognostic factors in resected NSCLC. Evaluating the influence of histological type and smoking habit on the immunological environment may lead to the establishment of immunological diagnosis and appropriate individualized immunotherapy for NSCLC.

Induction of antisporozoite antibodies by biting of transgenic Anopheles stephensi delivering malarial antigen via blood feeding.

We produced a transgenic mosquito expressing a rodent malaria vaccine candidate antigen in the salivary gland. Three tandemly repeated amino acid units from the repeat region of circumsporozoite protein of Plasmodium berghei (PbCS3R) fused to red fluorescent protein (monomeric DsRed) was chosen as a vaccine candidate antigen. Immunoblot and fluorescence microscopic analyses showed the transgene expression in the female salivary gland. The transgene product was released from the proboscis as a component of saliva. The monomeric DsRed-fusion expression system could be suitable for transgene secretion in the saliva of female mosquitoes. Mice repeatedly bitten by transgenic mosquitoes raised antibodies against P. berghei sporozoites, and the sera had protective ability against sporozoite invasion of human hepatoma HepG2 cells. These results suggest that transgene products are immunogenically active in saliva, and induce the antibodies to malaria parasite. These findings indicate that this technology has the potential for production of a 'flying vaccinator' for rodent malaria parasites.

Flying vaccinator; a transgenic mosquito delivers a Leishmania vaccine via blood feeding.

'Flying vaccinator' is the concept of using genetically engineered hematophagous insects to deliver vaccines. Here we show the generation of a transgenic anopheline mosquito that expresses the Leishmania vaccine candidate, SP15, fused to monomeric red fluorescent protein (mDsRed) in its salivary glands. Importantly, mice bitten repeatedly by the transgenic mosquitoes raised anti-SP15 antibodies, indicating delivery of SP15 via blood feeding with its immunogenicity intact. Thus, this technology makes possible the generation of transgenic mosquitoes that match the original concept of a 'flying vaccinator'. However, medical safety issues and concerns about informed consent mitigate the use of the 'flying vaccinator' as a method to deliver vaccines. We propose that this expression system could be applied to elucidate saliva-malaria sporozoite interactions.

Absence of IgD-CD27(+) memory B cell population in X-linked hyper-IgM syndrome.

The present study analyzed peripheral blood B cell populations separated by IgD and CD27 expression in six males with X-linked hyper-IgM syndrome (XHIM). Costimulation of mononuclear cells from most of the patients induced no to low levels of class switching from IgM to IgG and IgA with Staphylococcus aureus Cowan strain (SAC) plus IL-2 or anti-CD40 mAb (anti-CD40) plus IL-10. Measurable levels of IgE were secreted in some of the patients after stimulation with anti-CD40 plus IL-4. Costimulation with SAC plus IL-2 plus anti-CD40 plus IL-10 yielded secretion of significant levels of IgG in addition to IgM, but not IgA. The most striking finding was that peripheral blood B cells from all of the six patients were composed of only IgD+ CD27(-) and IgD+ CD27(+) B cells; IgD- CD27(+) memory B cells were greatly decreased. IgD+ CD27(+) B cells from an XHIM patient produced IgM predominantly. Our data indicate that the low response of IgG production in XHIM patients is due to reduced numbers of IgD- CD27(+) memory B cells. However, the IgG production can be induced by stimulation of immunoglobulin receptors and CD40 in cooperation with such cytokines as IL-2 and IL-10 in vitro.

The presence of phorbol ester responsive and non-responsive forms of the zeta isozyme of protein kinase C in mouse epidermal cells.

The possible involvement of zeta isozyme of protein kinase C (PKC zeta) in phorbol ester-induced signal transduction was investigated in mouse epidermal cells. Western blot analysis of RESOURCE Q column chromatography eluates obtained from 105,000 g supernatants and particulate fractions of epidermal cells was performed using anti-PKC zeta specific antibody. Anti-PKC zeta antibody recognised proteins in low salt range corresponding to 25-125 mM NaCl (low salt-eluted PKC zeta; 1-PKC zeta) as well as high salt range corresponding to 175-300 mM NaCl (high salt-eluted PKC zeta; h-PKC zeta) in both subcellular fractions. 1-PKC zeta and h-PKC zeta were detected as a doublet protein of 79,000 and 85,000 M(r) in 105,000 g supernatants, but as a 79,000 M(r) protein in particulate fractions. Immunoprecipitated 1-PKC zeta and h-PKC zeta with anti-PKC zeta specific antibody possessed phosphatidylserine (PS)-dependent protein kinase activity, but neither 1-PKC zeta nor h-PKC zeta were further activated by 40 nM phorbol 12-myristate 13-acetate (PMA) in the presence of PS. Furthermore, 1-PKC zeta and h-PKC zeta can be autophosphorylated, indicating that both 1-PKC zeta and h-PKC zeta are PKC zeta. Treatment of intact epidermal cells with PMA or other PKC activators caused the apparent shift of 79,000 M(r) 1-PKC zeta to the 85,000 M(r) from in particulate fractions. Prolonged treatment of the cells with PMA induced the downregulation of both forms of 1-PKC zeta in particulate fractions. Under the same condition, 1-PKC zeta in 105,000 g supernatants and h-PKC zeta in both fractions did not respond to PMA. This apparent shift was reversible and the content ratio of 85,000 to 75,000 M(r) 1-PKC zeta was decreased by acid phosphatase treatment, indicating that the apparent shift results at least in part from phosphorylation of 79,000 M(r) 1-PKC zeta. Total activity of 1-PKC zeta was increased in association with the apparent shift from the 79,000 to 85,000 M(r) form in response to PMA treatment of intact epidermal cells. All of these results indicate that PKC zeta is present as multiple forms in mouse epidermal cells, and that especially 1-PKC zeta in particulate fractions play a significant role(s) in PMA-induced signal transduction in mouse epidermal cells.

KP-10, a novel protein kinase C substrate in intact mouse epidermal cells, is phosphorylated by novel protein kinase C eta and/or zeta.

Recently this group found an endogenous substrate protein for Ca(2+)-independent novel protein kinase C (nPKC), i.e. KP-10 (pI 4.7/25,500 M(r)), in primary cultured mouse epidermal cells [Nishikawa, K. et al. (1992) Cell. Signal. 4, 757-776]. In the present study, the nPKC isozymes which phosphorylate KP-10 in these cells were determined. Western blot analysis revealed that PKC alpha, eta and zeta were present in epidermal cell 105,000 g supernatants and that the content of PKC zeta was much higher than those of PKC alpha and eta. Neither PKC beta, delta nor epsilon was detected in the 105,000 g supernatants. Phosphatidylserine and phorbol 12-myristate 13-acetate (PMA)-dependent KP-10 phosphorylating activity was immunoprecipitated by anti-PKC eta and zeta antibodies, but not by antiPKC alpha antibody. These results suggest that PKC eta and/or zeta phosphorylate KP-10 and play pivotal roles in intracellular signal pathways in intact epidermal cells.

Induction of protein kinase Czeta-related protein kinase by growth suppression in carcinogen-initiated epidermal cell-line WYF31 cells.

In primary cultured mouse epidermal cells, protein kinase C isozyme zeta (PKCzeta) consists of multiple forms, for example, low-salt eluted PKCzeta (1-PKCzeta; 79 and 85 kDa) and high-salt eluted PKCzeta (h-PKCzeta; 79 and 85 kDa) on anion-exchange column chromatography. In this study, biochemical and biophysical differences between 1-PKCzeta and h-PKCzeta were examined by using carcinogen-initiated mouse epidermal cell-line WYF31 cells, whose growth is stimulated by tumour promoter phorbol 12-myristate 13-acetate (PMA). The binding efficiency of h-PKCzeta to anti-PKCzeta antibody-affinity column was 10 times higher than that of 1-PKCzeta. T7-tagged rat PKCzeta overexpressed in WYF31 cells was recovered only in the high-salt eluted area on the anion-exchange column. Furthermore, when rat PKCzeta was stably overexpressed in WYF31 cells, the content of h-PKCzeta increased 4 to 5 times compared to that of parental cells, but the content of 1-PKCzeta was not altered. All of these results indicate that h-PKCzeta is the product of the PKCzeta gene (referred to as PKCzeta) and that 1-PKCzeta is closely related but different from PKCzeta (referred to as PKCzeta-related kinase). Interestingly, serum starvation of WYF31 cells caused a marked increase of the content of PKCzeta-related kinase with a concomitant decrease of PKCzeta content. These changes were reversed by stimulating the cell growth with 10% foetal calf serum. Prolonged treatment of starved cells with PMA, which induces the proliferation of WYF31 cells, also caused the downregulation of PKCzeta-related kinase. These results suggest that the expression levels of PKCzeta-related kinase and PKCzeta are differently regulated, and that the increased expression of PKCzeta-related kinase might play a significant role in the growth-suppression processes of WYF31 cells.

Effects of theophylline on human eosinophil functions: comparative study with neutrophil functions.

The understanding of theophylline as a bronchodilator has been reconsidered in recent years. We undertook to determine its immunomodulatory actions in granulocytes and elucidate their mechanism. Preincubation of neutrophils with theophylline (10(-5) to 5 x 10(-3) M) had a biphasic effect on O2(-) production stimulated with N-formyl-methionyl-leucyl-phenylalanine or C5a. Theophylline potentiates O2(-) production via adenosine A(2A) receptor antagonism induced by receptor-linked agonists from neutrophils, but not from eosinophils. The addition of theophylline caused a significant decline in neutrophil chemotaxis at lower concentrations than those for eosinophil motility. Theophylline reduces neutrophil chemotaxis via adenosine A1 receptor antagonism. At high concentrations, with an intracellular cAMP accumulation as a result of phosphodiesterase (PDE) inhibition, theophylline also exerts an inhibitory effect on the O2(-) production and chemotaxis of both types of cells. The difference in theophylline's effect on neutrophils and eosinophils appears to depend on the existence of specific adenosine receptors. Theophylline thus modulates granulocyte functions in association with specific adenosine receptor antagonism and cAMP-PDE inhibition.

Theophylline induces neutrophil apoptosis through adenosine A2A receptor antagonism.

This study was designed to determine whether theophylline would augment granulocyte apoptosis via a mechanism of adenosine A2A receptor antagonism. A selective adenosine A2 receptor agonist (CGS-21680, 1 microM) exhibited the most efficient potency for decreasing neutrophil apoptosis for 16 h from 63+/-5 to 19+/-4% (P < 0.001); it exerted poor and adverse effects on eosinophil survival. A selective protein kinase A inhibitor KT-5720 (10 microM) reversed the capacity of dibutyryl cAMP but not CGS-21680 to induce an inhibitory effect on neutrophil apoptosis, suggesting that occupancy of adenosine A2 receptors inhibit neutrophil apoptosis by a cAMP-independent mechanism. Theophylline derivatives show the following pattern of potency for inducing neutrophil apoptosis competing with CGS-21680: 8-phenyltheophylline = 8-p-sulfophenyltheophylline > theophylline >> enprofylline. This pattern is consistent with the affinity established for A2A receptors. Theophylline demonstrated an additive effect to that of anti-Fas antibody (CH11, 1 microg/mL) in inducing neutrophil apoptosis, but not to that of adenosine deaminase or KF-17837 (a selective A2 receptor antagonist; 1 microM), suggesting conflicting effects on the receptor antagonism. These findings suggest that theophylline has an immunomodulatory action on neutrophil apoptosis via a mechanism of A2A antagonism.

HA1077, a protein kinase inhibitor, inhibits calponin phosphorylation on Ser175 in porcine coronary artery.

Calponin is a thin filament-associated protein which has been implicated in the modulation of the contractile state of smooth muscle via its interaction with actin and inhibition of the actin-activated myosin Mg-ATPase. This inhibitory effect is alleviated by phosphorylation of calponin at Ser175 in vitro by protein kinase C. The issue of calponin phosphorylation in intact smooth muscle in response to agonists that activate protein kinase C is controversial. We have produced a monoclonal antibody that specifically recognizes calponin phosphorylated at Ser175 and used it to analyze calponin phosphorylation in porcine coronary arterial smooth muscle stimulated with prostaglandin F2alpha or phorbol 12,13-dibutylate (PDB). Calponin phosphorylation increased rapidly in response to prostaglandin F2alpha concomitant with the increase in tension. Calponin was then dephosphorylated while force was maintained. Tension development in response to PDB was significantly slower, but again calponin phosphorylation paralleled force development. In this case, calponin dephosphorylation was very slow, consistent with prolonged activation of protein kinase C. The protein kinase inhibitors, HA1077 (1-5-(isoquinoline sulfonyl)-homopiperazine HCl) and HA1100 (1-hydroxy HA1077; 1-(hydroxy-5-isoquinoline sulfonyl-homopiperazine), inhibited tension development and calponin phosphorylation in a concentration-dependent manner with similar ED50 values in response to prostaglandin F2alpha and PDB. These results support physiological roles for calponin in force development in smooth muscle in response to agonists which trigger protein kinase C activation and in the latch state, i.e., force maintenance at low energy cost. Furthermore, the vasodilator effect of HA1077 and HA1100 is more likely due to inhibition of protein kinase C than of myosin light chain kinase.

Selectivity of action of staurosporine on Ca2+ movements and contractions in vascular smooth muscles.

We examined the effects of staurosporine, a protein kinase inhibitor, on Ca2+ movements and contractions due to KCl and 12-deoxyphorbol 13-isobutyrate (DPB), which are thought to activate myosin light chain kinase and protein kinase C, respectively. In rabbit aortae, staurosporine inhibited contractions due to KCl (65.4 mM) and DPB (1 mu M) with IC50 values of 140.5 +/- 1.3 nM and 13.3 +/- 1.3 nM, respectively. Calphostin C, a putative inhibitor of protein kinase C, inhibited DPB-induced contraction with much less effect on the KCl-induced one. On the other hand, wortmannin, an inhibitor of myosin light chain kinase, was 4 times more potent on KCl-induced contraction than the DPB-induced one. Staurosporine at 100 nM decreased the rise in cytosolic Ca2+ due to KCl, whereas wortmannin did not affect it. In rabbit cerebral arteries permeabilized with beta-escin, staurosporine at 100 nM, but not 30 nM, inhibited Ca2+ -induced contraction in the presence of 1 mM ATP. The results indicate that staurosporine preferentially inhibits a contraction dependent on protein kinase C than that dependent on myosin light chain kinase in vascular smooth muscles. Its ability to inhibit KCl-induced contraction involves inhibition of voltage-dependent Ca2+ channels.

Plasma cell generation from B-lymphocytes via CD27/CD70 interaction.

To produce antibodies, the differentiation of B cells into antibody-secreting cells, plasma cells, is required. We describe that ligation of CD27, which belongs to the tumor necrosis factor receptor (TNFR) family and is a memory marker of B cells, yields crucial signals that positively control the entry of B cells into the pathway to plasma cells. The triggering via CD27 by CD27 ligand (CD70) on purified peripheral blood B cells yielded an increase in the number of plasma cells in the presence of interleukin-10 (IL-10). The differentiation into plasma cells by a combination of IL-10 and CD70-transfectants occurred in CD27+ B cells, but not in CD27- B cells. Moreover, the addition of IL-2 to the IL-10 and CD70-transfectants greatly induced the differentiation into plasma cells. In the presence of only IL-2, IL-4 or IL-6, CD70-transfectants did not promote the differentiation into plasma cells. On the other hand, CD40 signaling increased the expansion of a B cell pool from peripheral blood B cells primarily activated by IL-2, IL-10 and anti-CD40 mAb. These data demonstrate that CD27 ligand (CD70) is a key molecule to direct the differentiation of CD27+ memory B cells toward plasma cells in cooperation with IL-10.

[Effect of G-CSF (nartograstim) on neutropenia (leukopenia) induced by taxane in metastatic breast cancer--time-course changes in neutrophil and leukocyte counts].

Since 1997, we have used docetaxel and paclitaxel as the second-line and third-line chemotherapies against anthracycline-resistant metastatic breast cancer. However, these taxane compounds induced neutropenia and leukopenia, which may be reversed by G-CSF (Nartograstim). We thus examined the therapeutic efficacy of nartograstim for time-course changes in neutrophil and leukocyte counts in these patients. No difference was observed in neutrophil or leukocyte count whether the patient was treated with docetaxel or paclitaxel. Neutrophil and leukocyte counts reached a nadir on days 7 to 8 after administration. With a 5-6 day administration of nartograstim, neutrophil or leukocyte counts recovered by the second or third day after the nadir, indicating that the chemotherapy was given safely with nartograstim. In these same patients receiving a given treatment cycle, the number of days until reaching the nadir were almost identical for neutrophils and leukocytes; however, the duration of the nadir and the time to count recovery was significantly longer for neutrophils than for leukocytes. In the clinical setting, the parameter "leukocyte count" has been occasionally used for evaluation of the severity of myelosuppression, because the data is more readily available. However, at least during the nadir, the "neutrophil count" should be used as the parameter of choice.

Synergistic augmentative effect of interleukin-10 and CD27/CD70 interactions on B-cell immunoglobulin synthesis.

Interleukin-10 (IL-10) is a potent cytokine that regulates immunoglobulin synthesis by B cells. CD27/CD70 interactions by direct cell-to-cell contact are also needed to produce substantial amounts of immunoglobulin. We have investigated the effects of IL-10 and CD27/CD70 interactions on the immunoglobulin synthesis. In the presence of IL-10 stimulation, the production of IgG, IgM and IgA was increased synergistically by the addition of CD27 ligand (CD70)-transfectants in a dose-dependent manner, which was completely blocked by anti-CD70 monoclonal antibody. In contrast, CD70-transfectants additively enhanced the immunoglobulin production in the presence of IL-2, IL-4, or IL-6. The synergistic enhancement of the immunoglobulin production by IL-10 and CD70-transfectants was remarkable in highly purified CD27+ B cells, but there was no immunoglobulin production in CD27- B cells. Furthermore, by the addition of CD70-transfectants, the synthesis of IgG1, IgG2, IgG3 and IgG4 was also enhanced in the presence of IL-10. On the other hand, IL-10 diminished CD27 expression in B cells. B-cell proliferation was augmented by CD70-transfectants with IL-10 or IL-10 plus IL-2. The addition of IL-2 further augmented the immunoglobulin production which was synergistically enhanced by IL-10 and CD27 triggering. Taken together, the co-operative response to IL-10 and CD27/CD70 interactions regulates B-cell immunoglobulin production.

IL-10 enhances B-cell IgE synthesis by promoting differentiation into plasma cells, a process that is inhibited by CD27/CD70 interaction.

Interleukin-10 (IL-10) is a major regulatory cytokine of inflammatory responses that is considered to play an important role in specific immunotherapy. However, whether IL-10 enhances or inhibits B-cell IgE production has remained a matter of contention. To clarify the effect of IL-10 on IgE synthesis in the presence of IL-4 and CD40 signalling, we examined B-cell proliferation, germline epsilon transcripts and plasma cell differentiation. In addition, the effect of CD27 signalling on IgE synthesis in the presence of IL-10, IL-4 and CD40 signalling was investigated. IL-10 facilitated the production of IgE in mononuclear cells and highly purified B-cells, enhanced B-cell proliferation and, most importantly, promoted the generation of plasma cells. However, IL-10 did not enhance expression of germline epsilon transcripts. The addition of CD27 signalling through the use of CD32-CD27 ligand (CD70) double transfectants significantly diminished the B-cell proliferation, IgE synthesis and plasma cell differentiation enhanced by IL-10. IL-10 enhances B-cell IgE production by promoting differentiation into plasma cells. CD27/CD70 interactions under IL-10 and sufficient CD40 cosignalling exert the opposite effect on IgE synthesis. The results of this study indicate that precautions are critical when planning immunotherapy using IL-10 in IgE-related allergic diseases.

Phosphorylation of calponin by PKC is blocked by F-actin in vitro.

Polyclonal antibody against phosphorylated calponin was raised in rabbits by application of the peptide corresponding to residues 183-195 of calponin phosphorylated by protein kinase C. When calponin was incubated with protein kinase C, only free calponin was recognized by this antibody and calponin of native thin filament or that binding to F-actin did not. In experiments done using [gamma-32P] ATP, no radioactivity was detected except for free calponin. Calponin phosphorylation was suppressed in an actin dose-dependent manner and the phosphorylation of calponin was completely blocked when the actin molar ratio to calponin exceeded 10. These data suggest that phosphorylation of calponin by protein kinase C was apparently blocked by F-actin.

[Tissue distribution of CA-125 in female reproductive organs].

In order to elucidate the origin of elevated serum CA-125 values in patients with endometriosis, CA-125 content in the uterine endometrium, myometrium, leiomyoma, fallopian tube, ovary, adenomyotic tissue and endometriotic tissue, were measured by RIA. The CA-125 content in the normal endometrium was 334.7 +/- 39.1 U/mg-protein, and this was significantly (p less than 0.001) higher than that in the myometrium (66.1 +/- 8.6), cervical gland (131.7 +/- 24.2), fallopian tube (111.6 +/- 15.8) and ovary (64.6 +/- 15.6). Endometriotic tissue contained 217.5 +/- 23.1, and adenomyotic tissue contained 235.3 +/- 47.7. No significant difference was found between tissue from cervical and corporal malignancies of the uterus, but ovarian cancer tissue contained a high CA-125 level compared to normal ovarian tissue. The mean concentration of CA-125 in menstrual blood was 377.9 +/- 79.9 U/ml and was about 11 times as high as that seen in peripheral blood in the same donor. The fact that the endometrium contained the highest amount of CA-125 among normal tissues examined supports the hypothesis that externally developed endometrium is the primary source of elevated serum CA-125 in patients with endometriosis.

Activation of phorbol ester responsive form of protein kinase C zeta in association with Ca(2+)-induced differentiation of primary cultured mouse epidermal cells.

In primary cultured mouse epidermal cells, protein kinase C (PKC) zeta consists of multiple forms: a low salt-eluted PKC zeta (1-PKC zeta, 79 and 85 kDa) and a high salt-eluted PKC zeta (h-PKC zeta, 79 and 85 kDa) by anion-exchange column chromatography (K. Nishikawa et al., Cell. Signal. 7, 491-504, 1995). In the present study, PKC isozyme-specific responses during terminal differentiation of epidermal cells, which were induced by the increase of Ca(2+)-concentration in culture medium, were examined. After 24 hr-treatment with 1.8 mM Ca2+, 79-kDa 1-PKC zeta in the particulate fraction was apparently shifted to the 85-kDa form. The phosphatidylserine-dependent kinase activity of this 1-PKC zeta was increased in association with the shift. These results suggest the pivotal role of 1-PKC zeta in the particulate fraction in the Ca(2+)-induced epidermal cell differentiation processes.