Association of preoperative inflammation-based prognostic score with survival in patients undergoing salvage esophagectomy.

Salvage esophagectomy (SALV) is potentially beneficial for patients with residual or relapsed esophageal carcinoma after definitive chemoradiotherapy (dCRT), although preoperatively identifying good candidates for SALV remains difficult. We investigated the prognostic impacts of inflammatory and nutritional status in patients undergoing SALV after dCRT. Forty-seven SALV patients were retrospectively reviewed, of whom 46 (98%) had squamous cell carcinoma and 1 (2%) adenocarcinoma. Possible prognostic factors included patients' demographic data, physical status, blood chemistry profiles, and clinical/pathological tumor features. The Glasgow prognostic score (GPS) was derived from preoperative C-reactive protein (CRP) and albumin values. Thirty (64%), 11 (23%), and 6 (13%) patients were classified into the GPS 0, 1, and 2, respectively, groups. None of the possible prognostic factors showed significant correlations with GPS. Patients with GPS 0 had better outcomes than those with GPS 1 or GPS 2 (Median survivals: 37.8, 15.9, and 5.1 months, respectively, P < 0.001). In the multivariable Cox proportional hazards model, GPS 1 (HR 5.62, 95% CI 1.94-16.4, P = 0.002), GPS 2 (HR 9.10, 95% CI 2.60-31.8, P < 0.001), R1/2 resection (HR 16.3, 95% CI 3.62-86.7, P < 0.001) and incomplete response to dCRT (HR 3.53, 95% CI 1.12-12.5, P = 0.03) were all independent risk factors for a poor outcome. Preoperative GPS is potentially useful for predicting outcomes in esophageal cancer patients undergoing SALV.

Short-term outcomes of robotic radical esophagectomy for esophageal cancer by a nontransthoracic approach compared with conventional transthoracic surgery.

Transthoracic esophagectomy (TTE) is believed to have advantages for mediastinal lymphadenectomy in the treatment of resectable esophageal cancer despite its association with a greater incidence of pulmonary complications and postoperative mortality. Transhiatal esophagectomy is regarded as less invasive, though insufficient in terms of lymph node dissection. With the aim of achieving lymph dissection equivalent to that of TTE, we have developed a nontransthoracic esophagectomy (NTTE) procedure combining a video-assisted cervical approach for the upper mediastinum and a robot-assisted transhiatal approach for the middle and lower mediastinum. We prospectively studied 22 accumulated cases of NTTE and verified feasibility by analyzing perioperative and histopathological outcomes. We compared this group's short-term outcomes with outcomes of 139 equivalent esophageal cancer cases operated on at our institution by conventional TTE (TTE group). In the NTTE group, there were no procedure-related events and no midway conversions to the conventional surgery; the mean operation time was longer (median, 524 vs. 428 minutes); estimated blood loss did not differ significantly between the two groups (median, 385 mL vs. 490 mL); in the NTTE group, the postoperative hospital stay was shorter (median, 18 days vs. 24 days). No postoperative pneumonia occurred in the NTTE group. The frequencies of other major postoperative complications did not differ significantly, nor were there differences in the numbers of harvested mediastinal lymph nodes (median, 30 vs. 29) or in other histopathology findings. NTTE offers a new radical procedure for resection of esophageal cancer combining a cervical video-assisted approach and a transhiatal robotic approach. Although further accumulation of surgical cases is needed to corroborate these results, NTTE promises better prevention of pulmonary complications in the management of esophageal cancer.

An innovative masticatory efficiency test using odour intensity in the mouth as a target marker: a feasibility study.

A large number of studies have reported the findings for masticatory efficiency tests; however, some objective masticatory efficiency tests have a drawback, in that subjects are required to spit out the test material. This study examined the possibility of a masticatory efficiency test that evaluates the intensity of odours released when chewing an odour compound-containing material. A total of 20 volunteers were used in this study. The odour intensity in the breath after chewing a gum was measured by portable odour sensor device. The odour intensity after chewing the gum was measured over four chewing durations and at four intervals between spitting out and measurement of the odour intensity. The volume of stimulated saliva was measured by calculating the difference in the weight of the gauze before and after chewing to examine the effect of saliva flow. With an increase in chewing duration, odour intensity reduced. The odour intensity was the highest immediately after chewing. There was a positive correlation between odour intensity and gummy jelly-related masticatory efficiency test value (G-METV), which was significant for 10-s chewing. The regression equation was calculated from three objective variables of odour intensity and G-METV as dependent variable. Pearson's correlation coefficient between G-METV and the odour intensity-related masticatory efficiency value (O-METV) was 0·68. The coefficient of variation of O-METV was significantly lower than that of G-METV. These results suggest that the masticatory performance can be estimated by measuring the odour intensity immediately after chewing food containing odour compounds for 10 s.

The timing of worm exclusion in dogs repeatedly infected with the cestode Echinococcus multilocularis.

Experimental Echinococcus multilocularis infection and deworming was repeated three or five times in nine dogs at various re-infection schedules. The mean number of worms decreased more than 91% in dogs with repeated infection, compared to first infection controls (n= 6). The copro-antigen assay and the egg count in the faeces suggested that the worm burden gradually decreased each time the dogs were re-infected. To examine whether such worm exclusion was a non-specific response, five dogs were sequentially infected with the parasite four times and subsequently fed freely for 6 months. Even after the 6-month interval, the five dogs that were infected five times with the parasite were still able largely to exclude the adult worms. The results suggested that the ability of worm exclusion in dogs that developed a resistance did not become rapidly extinct. Observation of the condition of faeces and the excretion of hooks in the faeces of repeatedly infected dogs revealed that the exclusion of worms started at the first week after the re-infection, and it continued during the patent period. Serum antibodies specific to the parasite antigen increased gradually until the third infection and significantly decreased during the 6-month interval. There was little enhancement of serum antibodies after the fifth infection in most dogs, although no clear correlation was observed between the antibody response and the worm burden. These findings suggested the possibility of developing a vaccine.

Elevated liver enzymes are related to progression to impaired glucose tolerance in Japanese men.

AIMS: To investigate whether the elevation of liver enzymes is associated with the progression from normal to impaired glucose tolerance. METHODS: A historical cohort study was conducted in 594 male workers at public schools, who had normal glucose tolerance at baseline. The progression to impaired glucose tolerance and impaired fasting glycaemia during a mean follow-up of 3.1 years was measured using an oral glucose tolerance test. RESULTS: Overall, 141 (23.7%) subjects developed impaired glucose tolerance and 68 (11.4%) subjects developed impaired fasting glycaemia, 23 of whom had combined impaired fasting glycaemia/impaired glucose tolerance. The incidence of impaired glucose tolerance increased significantly with increasing quartiles of serum aspartate aminotransferase, alanine aminotransferase and γ-glutamyltransferase (P for trend <0.01). In Cox proportional hazards regression analysis, after adjusting for comprehensive risk factors, including plasma glucose levels, BMI and homeostatic model assessment of insulin resistance, the risk of progression to impaired glucose tolerance was significantly higher in the highest quartile of alanine aminotransferase than in the lowest quartile (hazard ratio 2.5; 95% CI 1.1-5.7). A significant association between alanine aminotransferase and the progression to impaired glucose tolerance was found after further adjustments for other liver enzymes or after the sample was limited to those with BMI < 25.0 kg/m(2) or with fasting plasma glucose < 5.5 mmol/l. CONCLUSIONS: A higher level of alanine aminotransferase was independently associated with progression from normal to impaired glucose tolerance in Japanese men. The elevation of alanine aminotransferase may be a change that occurs early in the evolution of diabetes.

Effect of reducing dietary forage in lower starch diets on performance, ruminal characteristics, and nutrient digestibility in lactating Holstein cows.

This experiment evaluated the effect of feeding a lower starch diet (21% of dry matter) with different amounts of forage (52, 47, 43, and 39% of dry matter) on lactational performance, chewing activity, ruminal fermentation and turnover, microbial N yield, and total-tract nutrient digestibility. Dietary forage consisted of a mixture of corn and haycrop silages, and as dietary forage content was reduced, chopped wheat straw (0-10% of dry matter) was added in an effort to maintain chewing activity. Dietary concentrate was adjusted (corn meal, nonforage fiber sources, and protein sources) to maintain similar amounts of starch and other carbohydrate and protein fractions among the diets. Sixteen lactating Holstein cows were used in replicated 4×4 Latin squares with 21-d periods. Dry matter intake increased while physically effective neutral detergent fiber (peNDF1.18) intake was reduced as forage content decreased from 52 to 39%. However, reducing dietary forage did not influence milk yield or composition, although we observed changes in dry matter intake. Time spent chewing, eating, and ruminating (expressed as minutes per day or as minutes per kilogram of NDF intake) were not affected by reducing dietary forage. However, addition of chopped wheat straw to the diets resulted in greater time spent chewing and eating per kilogram of peNDF1.18 consumed. Reducing dietary forage from 52 to 39% did not affect ruminal pH, ruminal digesta volume and mass, ruminal pool size of NDF or starch, ruminal digesta mat consistency, or microbial N yield. Ruminal acetate-to-propionate ratio was reduced, ruminal turnover rates of NDF and starch were greater, and total-tract digestibility of fiber diminished as dietary forage content decreased. Reducing the dietary forage content from 52 to 39% of dry matter, while increasing wheat straw inclusion to maintain chewing and rumen function, resulted in similar milk yield and composition although feed intake increased. With the lower starch diets in this short-term study, the minimal forage content to maintain lactational performance was between 39 and 43%.

Reduction of malaria transmission by transgenic mosquitoes expressing an antisporozoite antibody in their salivary glands.

We have previously developed a robust salivary gland-specific expression system in transgenic Anopheles stephensi mosquitoes. To establish transgenic mosquito lines refractory to Plasmodium falciparum using this system, we generated a transgenic mosquito harbouring the gene encoding an anti-P. falciparum circumsporozoite protein (PfCSP) single-chain antibody (scFv) fused to DsRed in a secretory form (mDsRed-2A10 scFv). Fluorescence microscopy showed that the mDsRed-2A10 scFv was localized in the secretory cavities and ducts of the salivary glands in a secreted form. To evaluate P. falciparum transmission-blocking in a rodent malaria model, a transgenic Plasmodium berghei line expressing PfCSP in place of PbCSP (PfCSP/Pb) was constructed. The PfCSP/Pb parasites were able to bind to the mDsRed-2A10 scFv in the salivary glands of the transgenic mosquitoes. Importantly, the infectivity of the transgenic mosquitoes to mice was strongly impaired, indicating that the parasites had been inactivated. These results suggest that salivary gland-specific expression of antisporozoite molecules could be a promising strategy for blocking malaria transmission to humans.

Visualization and live imaging analysis of a mosquito saliva protein in host animal skin using a transgenic mosquito with a secreted luciferase reporter system.

Mosquitoes inject saliva into a vertebrate host during blood feeding. The analysis of mosquito saliva in host skin is important for the elucidation of the inflammatory responses to mosquito bites, the development of antithrombotic drugs, and the transmission-blocking of vector-borne diseases. We produced transgenic Anopheles stephensi mosquitoes expressing the secretory luciferase protein (MetLuc) fused to a saliva protein (AAPP) in the salivary glands. The transgene product (AAPP-MetLuc) of transgenic mosquitoes exhibited both luciferase activity as a MetLuc and binding activity to collagen as an AAPP. The detection of luminescence in the skin of mice bitten by transgenic mosquitoes showed that AAPP-MetLuc was injected into the skin as a component of saliva via blood feeding. AAPP-MetLuc remained at the mosquito bite site in host skin with luciferase activity for at least 4 h after blood feeding. AAPP was also suspected of remaining at the site of injury caused by the mosquito bite and blocking platelet aggregation by binding to collagen. These results demonstrated the establishment of visualization and time-lapse analysis of mosquito saliva in living vertebrate host skin. This technique may facilitate the analysis of mosquito saliva after its injection into host skin, and the development of new drugs and disease control strategies.

Hepatotoxicity of sub-nanosized platinum particles in mice.

Nano-sized materials are widely used in consumer products, medical devices and engineered pharmaceuticals. Advances in nanotechnology have resulted in materials smaller than the nanoscale, but the biologic safety of the sub-nanosized materials has not been fully assessed. In this study, we evaluated the toxic effects of sub-nanosized platinum particles (snPt) in the mouse liver. After intravenous administration of snPt (15 mg/kg body weight) into mice, histological analysis revealed acute hepatic injury, and biochemical analysis showed increased levels of serum markers of liver injury and inflammatory cytokines. In contrast, administration of nano-sized platinum particles did not produce these abnormalities. Furthermore, snPt induced cytotoxicity when directly applied to primary hepatocytes. These data suggest that snPt have the potential to induce hepatotoxicity. These findings provide useful information on the further development of sub-nanosized materials.

Potential responders to FOLFOX therapy for colorectal cancer by Random Forests analysis.

BACKGROUND: Molecular characterisation using gene-expression profiling will undoubtedly improve the prediction of treatment responses, and ultimately, the clinical outcome of cancer patients. METHODS: To establish the procedures to identify responders to FOLFOX therapy, 83 colorectal cancer (CRC) patients including 42 responders and 41 non-responders were divided into training (54 patients) and test (29 patients) sets. Using Random Forests (RF) algorithm in the training set, predictor genes for FOLFOX therapy were identified, which were applied to test samples and sensitivity, specificity, and out-of-bag classification accuracy were calculated. RESULTS: In the training set, 22 of 27 responders (81.4% sensitivity) and 23 of 27 non-responders (85.1% specificity) were correctly classified. To improve the prediction model, we removed the outliers determined by RF, and the model could correctly classify 21 of 23 responders (91.3%) and 22 of 23 non-responders (95.6%) in the training set, and 80.0% sensitivity and 92.8% specificity, with an accuracy of 69.2% in 29 independent test samples. CONCLUSION: Random Forests on gene-expression data for CRC patients was effectively able to stratify responders to FOLFOX therapy with high accuracy, and use of pharmacogenomics in anticancer therapy is the first step in planning personalised therapy.

Protective effects of mineralocorticoid receptor blockade against neuropathy in experimental diabetic rats.

AIMS: Mineralocorticoid receptor (MR) blockade is an effective treatment for hypertension and diabetic nephropathy. There are no data on the effects of MR blockade on diabetic peripheral neuropathy (DPN). The aim of this study was to determine whether MRs are present in the peripheral nerves and to investigate the effectiveness of MR blockade on DPN in streptozotocin (STZ)-induced diabetic rats. METHODS: Expression of MR protein and messenger RNA (mRNA) was examined in the peripheral nerves using Western blot analysis and RT-PCR. We next studied the effects of the selective MR antagonist eplerenone and the angiotensin II receptor blocker candesartan on motor and sensory nerve conduction velocity (NCV), morphometric changes and cyclooxygenase-2 (COX-2) gene and NF-κB protein expression in the peripheral nerves of STZ-induced diabetic rats. RESULTS: Expression of MR protein and mRNA in peripheral nerves was equal to that in the kidney. Motor NCV was significantly improved by 8 weeks of treatment with either eplerenone (39.1 ± 1.2 m/s) or candesartan (46.4 ± 6.8 m/s) compared with control diabetic rats (33.7 ± 2.0 m/s) (p < 0.05). Sensory NCV was also improved by treatment with candesartan or eplerenone in diabetic rats. Eplerenone and candesartan caused significant improvement in mean myelin fibre area and mean myelin area compared with control diabetic rats (p < 0.05). COX-2 mRNA and NF-κB protein were significantly elevated in the peripheral nerves of diabetic rats compared with control rats, and treatment with eplerenone or candesartan reduced these changes in gene expression (p < 0.05). CONCLUSION: MR blockade may have neuroprotective effects on DPN.

Effect of surface charge on nano-sized silica particles-induced liver injury.

Nanomaterials are used frequently in microelectronics, cosmetics and sunscreen, and research for the development of nanomaterial-based drug delivery systems is promising. We previously reported that the intravenous administration of unmodified silica particles with a diameter of 70 nm (SP70) caused hepatic injury. Here, we examined the acute hepatic toxicity of SP70 modified with amino group (SP70-N) or carboxyl group (SP70-C). When administered intravenously into mice, SP70-N and SP70-C dose-dependently increased the serum level of alanine aminotransferase (ALT). However, the toxicity levels of surface charge-modified silica particles were much less weaker than the level of unmodified particles. When SP70 was repeatedly administered at 40 mg/kg twice a week for 4 weeks into mice, the hydroxyproline content of the liver significantly increased. Azan staining of the liver section indicated the extensive fibrosis. To the contrary, the repeated administration of SP70-N or SP70-C at 60 mg/kg twice a week for 4 weeks into mice did not cause the hepatic fibrosis. These findings suggest that the surface charge of nanomaterials could change their toxicity.

Hepatotoxicity of silica nanoparticles with a diameter of 100 nm.

Nanomaterials have potential toxicity that is not found in micromaterials, and it is therefore essential to understand their biological activity and potential toxicity. We focused on silica nanoparticles, since it was previously reported that the intravenous administration of silica nanoparticles with a diameter of 70 nm (SP70) causes hepatic injury. In the present study, we focused on the effects of the particle diameter of silica. We found that silica nanoparticles caused acute liver toxicity at a diameter of 100 nm, and that liver sinusoidal endothelial cells are directly involved in silica nanoparticle-induced liver injury. These findings suggest that the diameter of nanoparticles has great influence on silica nanoparticle-induced liver injury.

Effect of 70-nm silica particles on the toxicity of acetaminophen, tetracycline, trazodone, and 5-aminosalicylic acid in mice.

Exposure to nano-sized particles is increasing because they are used in a wide variety of industrial products, cosmetics, and pharmaceuticals. Some animal studies indicate that such nanomaterials may have some toxicity, but their synergistic actions on the adverse effects of drugs are not well understood. In this study, we investigated whether 70-nm silica particles (nSP70), which are widely used in cosmetics and drug delivery, affect the toxicity of a drug for inflammatory bowel disease (5-aminosalicylic acid), an antibiotic drug (tetracycline), an antidepressant drug (trazodone), and an antipyretic drug (acetaminophen) in mice. Co-administration of nSP70 with trazodone did not increase a biochemical marker of liver injury. In contrast, co-administration increased the hepatotoxicity of the other drugs. Co-administration of nSP70 and tetracycline was lethal. These findings indicate that evaluation of synergistic adverse effects is important for the application of nano-sized materials.

A toxicological evaluation of a claudin modulator, the C-terminal fragment of Clostridium perfringens enterotoxin, in mice.

Tight junctions (TJs) maintain cellular polarity between the apical and basolateral region of epithelial cells. Claudin, a tetra-transmembrane protein, plays a pivotal role in the barrier function of TJs. We previously found that a claudin modulator, the C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE), may be a promising candidate for improving the mucosal absorption of drugs. C-CPE is a fragment of enterotoxin, and putative CPE claudin receptors are highly expressed in liver and kidney. The safety and antigenicity of C-CPE must be evaluated for future clinical application. Therefore, we evaluated whether C-CPE administration in mice leads to tissue injury or production of antibodies. Intravenous administration of C-CPE at 5 mg/kg, which is a more than 25-fold higher dose than that used in a murine mucosal absorption model, did not increase biochemical markers of liver and kidney injury even after 11 injections once a week. Nasal C-CPE administration (2 mg/kg) once a week for 11 administrations also did not increase these biochemical markers, but 6 administrations of C-CPE resulted in elevation of C-CPE-specific serum IgG. These results indicate that development of a less antigenic claudin modulator will be essential for future clinical application of a C-CPE-based mucosal absorption enhancer.

Clinical Features of Cytotoxic Lesions of the Corpus Callosum Associated with Aneurysmal Subarachnoid Hemorrhage.

BACKGROUND AND PURPOSE: Patients with SAH due to a ruptured intracranial aneurysm occasionally show reversible high-signal lesions in the splenium of the corpus callosum on DWI. These lesions are called cytotoxic lesions of the corpus callosum. This study retrospectively reviewed cases of aneurysmal SAH and investigated clinical features of cytotoxic lesions of the corpus callosum associated with SAH. MATERIALS AND METHODS: Participants comprised 259 patients with aneurysmal SAH who had undergone curative treatment at our hospital. We examined the following items related to cytotoxic lesions of the corpus callosum: occurrence rate, timing of appearance and disappearance of the lesions, lesion size, aneurysm location, severity of SAH, treatment method, clinical course, and outcome. RESULTS: Among the 259 cases, DWI detected cytotoxic lesions of the corpus callosum in 33 patients (12.7%). The mean periods from the onset of SAH to detection and disappearance of cytotoxic lesions of the corpus callosum were 6.3 days (range, 0-25 days) and 35.7 days (range, 9-78 days), respectively. Cytotoxic lesions of the corpus callosum were classified into 2 types: a small type localized in the splenium in 26 cases (78.9%) and a large type spread along the ventricle in 7 cases (21.2%). The severity of SAH, coiling, hydrocephalus, and poor mRS score at discharge were significantly higher in the group with cytotoxic lesions of the corpus callosum. However, multivariate analysis did not identify cytotoxic lesions of the corpus callosum as a risk factor for poor outcome. CONCLUSIONS: Cytotoxic lesions of the corpus callosum appear at a frequency of 12.7% in patients with aneurysmal SAH. Cytotoxic lesions of the corpus callosum associated with SAH take several days to appear and subsequently resolve within about a month. Cytotoxic lesions of the corpus callosum were likely to occur in patients with high-grade SAH but did not represent a predictor of poor outcome.

Combined damage to the right hemispheric hand area in the primary motor and sensory area plays a critical role in motor hemineglect.

Direct evidence of the anatomical localization of brain function is provided by functional neurological changes during awake surgery combined with data from preoperative functional magnetic resonance imaging and diffusion tensor imaging studies. The goal of the present study was to analyze the etiology and mechanism of motor hemineglect using these techniques. Of 29 patients with brain tumors within and near the primary motor area (M1) in whom awake surgery was employed from April 2004 through March 2007, 2 patients evinced motor hemineglect of the left hand during awake surgery. The brain tumors in these 2 cases alone were located just beside the hand area of M1 and the primary sensory area (S1) in the right hemisphere. In case 1, the U fibers that connected the areas activated by hand clenching in M1 with S1 were compressed by the brain tumor. These results suggest that the combination of damage to the right hemispheric hand area in M1 and S1 plays a critical role in the development of motor hemineglect.

The safety of a mucosal vaccine using the C-terminal fragment of Clostridium perfringens enterotoxin.

The C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) is a claudin-4 binder. Very recently, we found that nasal immunization of mice with C-CPE-fused antigen activated antigen-specific humoral and mucosal immune responses and that the deletion of the claudin-4-binding domain attenuated the immune responses. C-CPE-fusion strategy may be useful for mucosal vaccination. C-CPE is a fragment of enterotoxin, and the safety of C-CPE-fused protein is very important for its future application. In the present study, we investigated whether C-CPE-fused antigen induces immune responses without mucosal injury by using ovalbumin (OVA) as a model antigen. Immunohistochemical analysis showed that claudin-4 was expressed in epithelial cell sheets bordering the nasal cavity. Nasal immunization with C-CPE-fused OVA dose-dependently elevated the OVA-specific serum IgG titer, which was 1000-fold greater than the titer achieved by immunization with OVA or a mixture of OVA and C-CPE at 5 microg of OVA. Nasal immunization with C-CPE-fused OVA (5 microg of OVA) activated Th1 and Th2 responses. Histological analysis showed no mucosal injury in the nasal cavity or nasal passage. C-CPE-fused OVA exhibited mucosal vaccination without mucosal injury. These findings indicate thatclaudin-4-targeting using C-CPE can be a potent strategy for mucosal vaccination.

Distinct effects of fatty acids on translocation of gamma- and epsilon-subspecies of protein kinase C.

Effects of fatty acids on translocation of the gamma- and epsilon-subspecies of protein kinase C (PKC) in living cells were investigated using their proteins fused with green fluorescent protein (GFP). gamma-PKC-GFP and epsilon-PKC-GFP predominated in the cytoplasm, but only a small amount of gamma-PKC-GFP was found in the nucleus. Except at a high concentration of linoleic acid, all the fatty acids examined induced the translocation of gamma-PKC-GFP from the cytoplasm to the plasma membrane within 30 s with a return to the cytoplasm in 3 min, but they had no effect on gamma-PKC-GFP in the nucleus. Arachidonic and linoleic acids induced slow translocation of epsilon-PKC-GFP from the cytoplasm to the perinuclear region, whereas the other fatty acids (except for palmitic acid) induced rapid translocation to the plasma membrane. The target site of the slower translocation of epsilon-PKC-GFP by arachidonic acid was identified as the Golgi network. The critical concentration of fatty acid that induced translocation varied among the 11 fatty acids tested. In general, a higher concentration was required to induce the translocation of epsilon-PKC-GFP than that of gamma-PKC-GFP, the exceptions being tridecanoic acid, linoleic acid, and arachidonic acid. Furthermore, arachidonic acid and the diacylglycerol analogue (DiC8) had synergistic effects on the translocation of gamma-PKC-GFP. Simultaneous application of arachidonic acid (25 MicroM) and DiC8 (10 microM) elicited a slow, irreversible translocation of gamma-PKC- GFP from the cytoplasm to the plasma membrane after rapid, reversible translocation, but a single application of arachidonic acid or DiC8 at the same concentration induced no translocation. These findings confirm the involvement of fatty acids in the translocation of gamma- and epsilon-PKC, and they also indicate that each subspecies has a specific targeting mechanism that depends on the extracellular signals and that a combination of intracellular activators alters the target site of PKCs.